The addition of excess Tb 3؉ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum. Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen. The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium. The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (k ET ϳ360 and 120 s ؊1) from the triplet state of tryptophan to terbium followed by terbium decay. This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp 109 ) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp 109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp 109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9 -10 Å.Metalloenzyme research has been greatly aided by the isomorphic replacement of intrinsic and spectroscopically silent metals (i.e. Ca . Because of its similar size and preference for strong oxygen donor groups as ligands, the use of the extrinsic luminescent probe Tb 3ϩ as a replacement for Ca 2ϩ is particularly well established (1-4). For the typical concentrations used in terbium-protein systems, Ͻ10 Ϫ3 M, the emission of free terbium in solution is generally not observed following direct UV excitation with conventional sources because of its low extinction coefficient (⑀ 285 Ϸ0.4 M Ϫ1 cm Ϫ1 for Tb 3ϩ complexes of diethylenetriaminopentaacetic acid (5)), whereas, when bound to a protein and in proximity to photoexcited aromatic residues, energy transfer is very efficient (3). The enhanced terbium luminescence thus observed has been used analytically to determine binding constants (6, 7) and, most importantly, assuming that a Förster-type dipoledipole energy transfer mechanism is established, to extract intraprotein donor-acceptor pair distances (for example see Refs. 8 and 9). Although enhanced terbium luminescence has found extensive use in metalloenzyme studies, the nature of the energy transfer mechanism and the identification of the molecular donor states involved is far from clear. For some terbiumsubstituted metalloenzymes, energy transfer has been convincingly established to proceed by way of a long-range nonradiative transfer from protein aromatic singlet states (5, 10). At shorter donor-acceptor distances, however, a Dexter exchange mechanism is suggested (2). The observation of inc...

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.

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Circularly polarized luminescence (CPL) spectroscopy provides information on the excited-state chirality of a lumiphore analogous but complementary to information regarding the ground-state chirality derived from circular dichroism. While CPL spectra are sensitive to the structure surrounding the lumiphore, their applicability to studies of complex biomolecules is limited by the fact that as a rule these molecules contain several identical lumiphores in different environments (e.g., tryptophan residues in a protein). Moreover, the ensemble of biomolecules in solution frequently exists in an equilibrium among different conformational states that interconvert on a time scale longer than that involved in the emission of luminescence. Hence, a typical CPL spectrum is a superposition of several independent contributions with a high degree of spectral overlap. One effective approach for the resolution of such composite spectra is by monitoring the time evolution of the CPL signal and the luminescence decay after electronic excitation of the lumiphore and correlating the CPL components to those obtained from analysis of the luminescence decay. Individual terms in the CPL may then be assigned distinct decay components. Time-dependent CPL may also arise from excited-state interactions of the lumiphore that affect the local chiral properties during the excited state lifetime. In such cases each lumiphore may possess a timedependent CPL, and the time-resolved CPL (TR-CPL) then can be used to obtain information on the dynamics of excitedstate interactions.We have recently utilized time-resolved optical activity and demonstrated that we could assign each of the four components obtained in the analysis of the decay of the roomtemperature phosphorescence of bacterial glucose-6-phosphate dehydrogenase (G6PDH) with a time-independent gem derived from the analysis of the time-resolved circularly polarized phosphorescence from the enzyme's tryptophan residues (4). This assignment allowed us to conclude that the room-temperature phosphorescence of G6PDH originates in three or four tryptophan residues, each with a unique, timeindependent CPL contribution.In